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Addgene

Expression scaffold for dCas9+sgRNA
(Plasmid #171799)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 171799 Standard format: Plasmid sent in bacteria as agar stab 1 $85

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    TOPO
  • Vector type
    CRISPR

Growth in Bacteria

  • Bacterial Resistance(s)
    Kanamycin, 50 μg/mL
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    PICASSO: expression scaffold for proper dCas9-fusion + sgRNA expression
  • Insert Size (bp)
    1191

Resource Information

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

Encodes expression scaffold for proper dCas9-fusion and sgRNA co-expression. To be introduced in second subcloning step for PICASSO library preparation.

Insert encodes C-terminal dCas9-fusion epitope tag HA, stop codon, translational termination, chloramphenicol resistance marker, and sgRNA promoter.

Recommended primers for amplification (introduce HA+6His C-terminal epitope tags and T7 promoter for sgRNA expression):
5'-agtcagtcGTCGACTACCCGTACGACGTTCCGGACTACGCGggtggtcaccatcatcaccatcattaataagg-3'
5'-CAACCAACACTAGTCTATAGTGAGTCGTATTAGATCTTTAGGACGTCGATATCTG-3'

After amplification, the amplicon should be digested with SalI/SpeI for insertion into the dCas9+sgRNA paired expression vector (second subcloning step after peptide+sgRNA spacer insertion with internal SalI/SpeI restriction sites).

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    Expression scaffold for dCas9+sgRNA was a gift from Stephen Elledge (Addgene plasmid # 171799 ; http://n2t.net/addgene:171799 ; RRID:Addgene_171799)
  • For your References section:

    CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays. Barber KW, Shrock E, Elledge SJ. Mol Cell. 2021 Aug 4. pii: S1097-2765(21)00596-7. doi: 10.1016/j.molcel.2021.07.027. 10.1016/j.molcel.2021.07.027 PubMed 34390675